DK151398B - DEVICE FOR IMMUNOLOGICAL DETERMINATION OF ANTIGEN OR ANTIBODIES AND USE OF THIS DEVICE - Google Patents

Description

in 151398

The present invention relates to an apparatus for immunologically determining antigens or antibodies in human body fluids with a microtiter plate having apertures for recording samples to be examined and with solid carriers which can be charged with a complexing agent.

Such an apparatus is disclosed in DE publication no.

2530678. With this apparatus, the spherical coated carriers are placed in the openings in a plate of a tubular sphere holder. The determination of spherical carriers is complicated because the spheres must be washed separately for which cumbersome apparatus is required.

In addition, spherical supports have a relatively small surface area and therefore a low efficiency.

In another apparatus for carrying out radioimmunological methods known from DE Publication No. 2,262,479, cone-shaped carriers which can only be used with a handle are proposed. Since these carriers and the handle are moved during the washing and are also used together with the handle in the measuring apparatus, there is a particularly frequent risk of contamination.

The object of the invention is to provide an apparatus by which many carriers exhibiting a large surface can be easily handled and with which the danger of contamination is largely excluded.

The apparatus according to the invention is characterized by the characterizing part of claim 1.

0.5

The invention further relates to the use of the apparatus for various determinations.

For detection of hepatitis B surface antigen (HB AG) and the corresponding - 0 antibody (anti HB AB) by means of radioimmunoassays,

S

currently the following methods: 1. Ausria I, Ausria II, Ausab (Abbott Laboratories). These work according to the so-called sandwich principle.

> 5 The Ausria I method uses a small tube loaded with antibody in which the sample to be examined is filled. After incubation, in the presence of antigen, a complex antibody antigen (AB-AG) is formed, the antibody component being fixed to the small tube. The liquid is then removed, the tube washed, and a solution of radiolabelled 50 antibody is added. After yet another incubation, a complex antibody-antigen-labeled antibody (AB-AG-AB) is formed. The small tube is washed again and the radioactivity of the AB-AG-AB * complex (sandwich complex) is determined. The antibody is labeled by incorporation of radioactive iodine in known manner. The Ausria II method uses spherical solid supports instead of small tubes of J5. The Ausab method is very similar to the Ausria method. Here, however, a reverse structure of the sandwich complex, namely AG-AB-AG *, is chosen.

3 151398 2, Electro Nucleonics Laboratories' Riausure Method.

In this method, in the same way as in the Ausria II method, a sandwich complex is formed, but instead of spheres glass particles are used as solid supports.

5 3. Combi RIA Biotest Method. Upon this, a set antibody is added to the sample to be tested for antigen and the mixture is transferred to a small dish coated with antigen. After incubation and washing, radioactive antigen is added, incubated and washed again.

This is also one of the sandwich methods which requires cumbersome washing and separation operations.

With a low antigen content in the samples, all of these methods are inaccurate.

Use of the apparatus of the invention for the determination of antigens, especially hepatitis B surface antigen (HBgAG) or of antibodies (anti HBgAB), in human body fluids, especially human plasma, human serum and fractions thereof, by forming an antigen-antibody complex, in which antibody component is radiolabelled, 2q is characterized in that a solution of the radiolabelled antibody is added to the sample to be tested for antigen or antibody and then a carrier with unlabelled antigen loaded solid is contacted with the test liquid, after which the radioactivity of the carrier is measured after washing the carrier.

25

This avoids the disadvantages described above and the accuracy of the determination is increased, as the assessment is by direct measurement of the radioactivity on the solid phase, not by measuring the radioactivity of the above liquid, as was the case in the previously known radioimmunoassay. Apart from the labor savings of avoiding centrifugation and pipetting, the highest numbers are obtained in the area of the negative or very weak positive samples, which is a significant improvement and assurance of the test result.

In the case where the test sample contains antigen, 35 µ react with the radiolabeled antibody. At the subsequent step by which the carrier labeled with unlabeled antigen is immersed in reaction __ 1_ 1 Ί .... J> 9 λ X wiiwmI rA / 9 A «4 · <ί Μ X Λ · Ρ A · Am 4 lrlr L ΚΙ ΛΎ »Τ AVlnW 1 Λ · * Γ 4 151398 of the sample antigen content, react with the antigen adsorbed to the carrier. Thus, after washing the carrier, some degree of bound radioactivity which is inversely proportional to the antigen content of the sample will be found.

In the case where the test sample does not contain antigen but antibody, the antibody content in the sample fluid is increased. Thus, during the first incubation, no reaction occurs. Then, in the next step, contacting the sample liquid loaded with unlabelled antigen, a competition between the unlabelled and the labeled antibody in the reaction with the adsorbed antigen on the vehicle, which means that in this case a particular degree of radioactivity on the washed carrier, which in turn is inversely proportional to the antis tof content of the sample under study. If, in a series of tests, a decrease in radioactivity compared to a control sample containing neither antigen nor antibody was found, it is known that the sample in question contained either antigen or antibody, but it is not yet known whether is one or the other. To find out now, a so-called dif-Q ferential diagnosis is performed, in which another sample of the same kind is brought into contact with another carrier and incubated. The carrier is washed and brought into contact with radiolabeled antibody in an additional step and incubated. If this measurement of radioactivity is found to have an elevated number, it means that there was no antibody in the sample but antigen because the entire adsorbed antigen was consumed to form the complex antigen antibody. On the other hand, if a diminished number is found, it means that the sample antibody was used in the incubation with the adsorbed antigen to form the antigen-antibody complex, and that only few binding sites were free of the labeled antibody on the antigen-loaded carrier.

0

Preferably, a heparin stabilized, radiolabelled antibody is used.

Conveniently, the incubations are performed at a temperature of between 18. and 45 ° C for a time of 30 minutes to 15 hours, preferably at 45 ° C for 60 minutes.

5 151398

The apparatus of the invention is particularly suitable for the determination of HBgAG and antiHBgAB. It can in principle be used to detect all antigens against which specific antibodies can be developed. Also suitable. The apparatus is for the detection of antibodies whose 5 antigens are proteins or polypeptides and which can be prepared in purified form. It can also be used, such as the system HB AG /

S

antiHBgAB shows, to the relatively rare case, that in patient sera both antigen and antibody are present. Other uses of the apparatus of the invention lie in the determination of viral antigens and in the determination of blood clotting factors, especially factor VIII proteins.

BRIEF DESCRIPTION OF THE DRAWINGS An apparatus according to the invention is illustrated in more detail in the drawing. 1 is a perspective view and FIG. 2 is a frontal view 15 of the carrier. Pig. 3 is a measurement curve obtained scan described in the following section. 2nd

The apparatus has a microtiter plate 1 which has several rows of e.g. twelve openings 2, 2 ', 2 "... for examination of the specimens. Furthermore, the apparatus includes holding strips 3 »to which a number of 2o numbered small plates 4, 4 ', 4” ... belong, corresponding to the number of openings in a row in the microtiter plate. The small plates 4, 4 ', 4, f ... are attached to the retaining strip 3 by means of neck pieces 5, 5', 5 ".. ·» whose cross-section is smaller than the cross-section of the small plates. These neck pieces form fracture sites. After the small plates have reacted with the liquid to be examined in the openings in the microtiter plate, the small plates are broken off and dropped into small counter tubes 6, as indicated in Figure 2.

The following examples are intended to further illustrate the use of the apparatus of the invention.

Example 1: Determination of HB AG and antiHB AB.

S s

In this study, in the sample of a human body fluid to be examined may contain HB AG or antiHB AB or 35 ss neither of the two substances. In a first study, 0.2 ml of the material to be examined is incubated approx. 60 minutes at 45 ° C in a heating cabinet with the purified labeled antiHB AB (0.1 ml contains about 0.1 mcCi) in a microtiter plate, simultaneously carrying two HBgAGr positive, three HBgAG / antiHBsAB negative and seven HBgAG - weak positive control samples. After this incubation, the HBgAG-coated, comb-like carriers are introduced into the pre-incubated mixtures and further incubated at 45 ° C for 90 to 120 minutes. Then, the comb-like carriers are taken from the sample solutions and washed in a tub of tap water. The individual small test plates on the comb-like carriers are transferred to the small test tubes upon interruption at the fracture sites, and in a gamma-spectrum network the bound radioactivity on the individual small plates is detected. The following values were found on the control samples 1 to 12 and the LO samples 13 to 24 examined:

Try Time in min. r-r. \ (Pulses per mm.) L5 1 1.00 684 2 1.00 672 3 1.00 4644 4 1.00 4148 5 1.00 4087 20 6 1.00 1974 7 1.00 1997 8 1.00 1846 9 1.00 2035 10 1.00 1942 25 11 1.00 2455 12 1.00 1598 13 1.00 433 positive 14 1.00 340 positive 15 1.00 4996 30 16 1.00 4020 17 1.00 2946 18 1.00 2673 19 1.00 2445 20 1.00 2822 35 21 1.00 1317 positive 22 1.00 I55O positive 23 1.00 3991 24 1.00 4052 7 151398

Samples 1 and 2 are strongly HB AG positive control serum, samples 3 to 5: HB AG / antiKB AB negative control serum, and samples 6 to 12 are HB AG weak positive control serum. Of these three groups, the mean values were found, and for the seven HBAG low-positive control sera 5, 20% were added to the mean value, respectively.

At least six of the seven samples are now required to be within these established limits. If a sample is outside the two limit values (in the sample number 11 present here), its value is not taken into account in the evaluation. The average value of the 10 remaining six samples plus 20% applies as a limit value. Thus, each sample which has a lower numerical value than this limit value must be considered positive and is designated as positive in the table (samples 13, 14, 21 and 22).

After this first test, it must be determined whether the positive reaction is from HBgAG or from antiHBgAB. To make the differential determination, you now perform another experiment as follows:

Samples of each 0.2 ml are placed in the microtiter plate and incubated for 60 minutes at 45 ° C with comb-like supports coated with HB AG. The carriers are then removed from the sample solutions as described above, washed and further incubated for 60 to 120 minutes in 0.2 ml of 12 * 5 in a 1 plus 1 dilution of the labeled antiHB_AB in physiological saline. Then, the comb-like supports are washed again and examined, as described above, in a gamma-spectrum network. It should be noted here that in the first sample series, in the presence of HB AG in the sample, a specific inhibition of the radiolabeled antibody occurs, in the presence of antiHBoAB in the sample only an increase of the total antibody amount in the reaction mixture. After insertion of the comb-like carriers, the non-bound, radiolabeled antibody bound to the sample antigen is bound to the surface of the small sample plates and, in the presence of HB AB, both a portion of the sample antibody and a portion of the radiolabeled antibody are fixed on the surface of the comb-like carriers.

35 Compared to HB AG / antiHB AB negative control samples, both the presence of HB AG in a sample and the presence of HBgAB in a sample result in a reduction of the numerical values on the small sample plates. In the differential assay, no immunological reaction occurs by incubating the comb-like carriers with the samples in the presence of HB AG in the samples or with samples that are HB AG /

U.S

OY \ * f “1 Hti Λ" Π Μ ΩΓΡΛ 4 * T 1 Tft XJtr · ΐ n rvvi Λν »« ιλ ·! · 4 ITD AD 4 w.iAIr «vt .4 λ 4- 1% / \« <ν \ 4 · 4 · Λ 8 The antigen binding sites on the comb-like carriers.

If the comb-like carriers are now washed and re-incubated, this time with 125 labeled antiHB AB, the radioactive material can occupy only the s 3 yet free antigen-binding sites on the solid phase. Thus, in the presence of antiHBsAB in the samples, the numerical values are reduced. In the presence of HB AG or HB AG / antiHB AB-

S SS

negative samples remain the number values large.

The results found are listed in the following table:

Try Time in min. (Pulses per minute) 1 1.00 3866 15 2 1.00 3716 3 1.00 3949 4 1.00 3799 5 1.00 3964 6 1.00 1435 20 7 1.00 1504 8 1.00 1605 9 1.00 1627 10 1.00 1482 11 1.00 1449 25 12 1.00 1448

13 1.00 3951 positive HBgAG

14 1.00 4026 positive HBgAG

15 1.00 3910 16 1.00 3996 30 17 1.00 3884 18 1.00 3843 19 1.00 3917 20 1.00 3871

21 1.00 363 positive aifcLHB AB

35 s

22 1.00 1335 positive ardHBsAB

23 1.00 3776 24 1.00 3778 9 151398

For control sera # 1 and 2 HB AG, slightly positive serum was used.

u

No. 3 to 5 are HBAG / antiHB AB negative sera and the control samples S s # 6 to 12 are antiHB AB positive sera. Again, as above c, the average values for these three groups and the limit value for the antiHB AB positive group were found. Each sample whose numerical value is below this limit value should be considered positive for antiHB AB (samples 21, 22). Samples 13 and 14 gave high numerical values and should be judged as HB AG positive. All other tests, as in both

ISLAND

reactions have given high numerical values are HB AG and antiHB AB negative.

S s .o

Example 2: Determination of alpha-fetoprotein (AFP).

Alpha-fetoprotein, which was purified by gel chromatography, is used to coat the comb-like carriers. Anti-alpha-fetoprotein-5 antibody is purified via an immunosorbent (BRCN sepharose) and labeled with

The normal value is between 0 and 40 ng / ml serum. In certain liver diseases and in carcinomas the values are increased to 100 ng / ml to 1000 ng / ml.

The quantitative determination of the antigens or antibodies, respectively, is carried out as follows: Instead of the control sera, a dilution series of the antigen to be determined is carried out. The dilution rows for preparation of a measurement curve are made as follows: Preferably, geo-metric dilutions of the standard in negative control serum are selected and additional negative control serum and the labeled antibody alone are used as control samples. The pure antibody samples provide values for the maximum binding capacity of the material. The negative i0 control serum samples give maximum binding values in the presence of negative samples. With increasing concentration of the standard antigen in the measurement curve, the bound activity decreases proportionally with the concentration. All control samples must be subjected to at least a double determination, preferably a triple determination.

One of the following single values measured curve is shown in the diagram (Fig. 3) and denoted by AFP standard: 35 10 151398

Single value is Measurement curve 10 ng AFP ¢ ^ = 2516 02 = 2488 c = 2460 cpm c3 = 2376 20 ng c1 = 2276 0 ^ = 2360 c = 2287 cpm c3 = 2225 40 ng 0 -, = 2100 02 = 2045 c = 2100 cpm c3 = 2156 80 ng ο1 = 1673 02 = 1515 c = 1581 cpm c3 = 1555 160 ng c ± = 983 c2 = 875 c = 955 cpm c3 = 1006 320 ng AFP c ± = 614 C2 = 588 c = 567 cpm c3 = 499 640 ng AFP 0 - ^ = 143 C2 = 205 c = 171 cpm c3 = 166 1280 ng c; L = 122 C2 = 166 c = 126 cpm c3 = 89 negative control serum ο1 = 2625 02 = 2783 c = 2707 cpm c3 = 2712 125 max. binding with J antibody c1 = 3540 02 = 3236 c = 3340 cpm c3 = 3245 Π 151398

The further procedure is now the same as in Example 1 of the HBgAG sample.

The samples to be examined are incubated with the labeled antiserum and then the comb-like carriers coated with AFP are inserted into the microtiter plate. After a further incubation, the comb-like carriers are washed and the activity recorded on the individual small sample plates in a gamma spectrometer. The numerical values found were as follows: 0

Sample Sample 1 0- ^ 2525 4 c- ^ 146 c2 = 2481 c2 = 135 c ^ = 24ll c = 2472 cpm c ^ = 104 c = 128 cpm 2 02 = 1142 5 0 ^ = 2017 c2 = 1276 c2 = 1965 0 - ^ = 1217 c = 1212 cpm 0 - ^ = 2034 c = 2005 cpm O 3 c1 = 475 c2 = 512 c ^ = 489 c = 492 cpm

These were entered on the measurement chart and gave the following finding: 5

Sample 1 negative 2 140 - 145 ng 3 340 ng 0 4 approx. 1000 ng, 5 48 ng

Example 3: Determination of HCG (human chorionic gonadotropin).

5 HCG, a glycoprotein hormone, is used to coat the comb-like 125 carriers. AntiHCG AB is labeled with J radioactive. The normal value is at 2mIU / ml. When carcinomas occur, the values are increased to 100 mIU / ml. The determination of HCG is particularly valuable in the follow-up of chemotherapy.

i2 151398

Example 4: Determination of gastrin.

Gastrin is used to coat the comb-like carriers. As an antibody solution, a J-labeled antigastrin AB solution is prepared.

5 The determination of gastrin is of growing importance as it is produced directly by tumors. Furthermore, elevated gastrin concentration occurs in the absence of gastric acid secretion.

Determination of antibodies.

10

Example 5: Determination of IgE.

IgE is a specific immunoglobulin whose presence may indicate clues to atopic allergies. In this case, on the 15 comb-like carriers, an antigen that acts as an antigen is bound. If the samples are now preincubated with a labeled IgE and the carriers are then inserted, the bound activity of the carriers is inversely proportional to the IgE concentration in the study material.

Example 6: Determination of allergen.

If, instead of anti-IgE, a specific allergen (e.g., penicillin) is coupled to the comb-like carriers, the same assay method can be performed allergen-specific by labeled antibodies against this allergen. 25

Example 7: Determination of tetanus antibody.

Tetanus antibodies bind to the comb-like carriers. The pre-inku-125 samples are prepared with labeled tetanus toxin and then the comb-like carriers are inserted. Presence of tetanus antibodies in the sample is detected by decreasing the number values compared to the control sample.

Example 8: Determination of DNS antibody.

35

The determination of anti-DNS antibody titers is a sensitive method for the detection of lupus erythematodes visceralis and enables differential determination against collagenoses of second genesis. The comb-like supports 125 are coated with anti-DNS antibody. The J-labeled DNS is incubated with the 40 sample and then the comb-like carriers are inserted. The presence of anti-DNS antibodies is expressed by diminished number values on them

Claims (9)

13 151398 small sample plates. Example 9: Determination of Immune Complexes. Method a) The comb-like carriers are coated with heat-aggregated IgG (IgG is aggregated by incubation at 60 ° C after 20 minutes). This 125 material behaves immunologically as immune complexes. The J-labeled iheum factor is preincubated with the sample and binds to immune complexes. Then the comb-like carriers are inserted. The unused tracer binds to the small sample plates. Lower numerical values on the sample plates 0 indicate the presence of immune complexes in the sample. Method b) The J-labeled iheumaf actor can be replaced with the J-labeled Clq (first component). 5 The determination of antigen-antibody complexes is gaining importance as these complexes are linked to the pathogenesis of a variety of disease images, such as arthritis, systemic lupus erythematosus, glomerulonephritis, hepatitis and possibly cancer. Example 10: Co-determination of antigen and antibody The presence of antibodies against the antigen to be assayed is unusual in ordinary radioimmunoassay, since mostly very small antigens are determined (e.g. hormones) which by themselves do not appear antigenic or which the body's own substances do not produce antibodies. However, since high molecular pathogenic substances are also being investigated using these methods, occasional occurrence of antibodies against these substances in individual samples must be considered. 10 Patent Claims. 1 An apparatus for immunologically determining antigens or antibodies in human body fluids having a microtiter plate with openings for recording samples to be examined and with solid supports which can be charged with a complexer, characterized in that the carriers consist of small plates ( 4, 4 ', 4 "...) or pins attached to a retaining strip (3) and can be inserted into the openings (2, 2', 2" ...) of the microtiter plate (1), between which the holding strip (3) and each of the individual small plates (4, 4 ', 4 "...) there is a groove for forming a breaking point. Apparatus according to claim 1, characterized in that small plastic support plates (4, 4 ', 4' ...) are used. Apparatus according to claim 1 or 2, characterized in that the carrier is loaded with antigen by immersion in an antigen solution for a time from 10 to 24 hours. Use of an apparatus according to claim 1 for the determination of antigens, in particular hepatitis B surface antigen (HB AG) or of antibodies (anti HB AB), in human body fluids, especially human plasma, human S serum and fractions thereof. forming an antigen-antibody complex in which the antibody component is radiolabelled, characterized in that a solution of the radiolabelled antibody is loaded into the sample to be tested and an unlabelled antigen loaded carrier then is brought into contact with the sample liquid and the radioactivity of the carrier is measured after washing the carrier. Use according to claim 4, characterized in that a heparin stabilized, radiolabelled antibody is used. 25 Use according to claim 4 or 5, characterized in that the incubation is carried out at a temperature between 18 and 45 ° C for a time from 30 minutes to 15 hours, preferably at 45 ° C for 6 minutes. Use according to claim 4 for the determination of an antigen, characterized in that a solution of a radiolabeled antibody corresponding to the antigen is added to a sample of the human body fluid and incubated at a temperature between 18 and 45 ° C. 30 minutes to 15 hours, forming an antigen-radioactive anti-OC * 00 substance complex (AG-AB complex), after which a carrier labeled with unlabelled antigen is contacted with the sample and incubated at a temperature between 18 and 45 ° C for 30 minutes to 15 hours after which the carrier is washed, and the radioactivity of the radioactivity labeled on the carrier adsorbed is measured, whereby the measured radioactivity is inversely proportional to the antigen content of the sample in the presence of antigen in the sample. Use according to claim 4 for the determination of an antibody, characterized in that a solution of a radiolabeled antibody is added to a sample of the human body fluid and incubated at a temperature between 18 and 45 ° C for 30 minutes to 15 hours, an unlabeled antigen-loaded carrier is contacted with the sample and incubated at a temperature between 18 and 45 ° C for 30 minutes to 15 hours, thereby forming, upon adsorption on the carrier, an antibody-anti-gene complex (AB -AG complex) and a radioactive antibody-antigen complex (AB-AG complex) upon which the carrier is washed and the radioactivity of the radioactively labeled antibody adsorbed on the carrier is measured, thereby measuring the measured radioactivity inversely proportional to the antibody content of the sample in the presence of antibody in the sample. Use according to claim 7 for determining whether in a sample of human body fluid there is hepatitis antigen (HB AG) or hepatitis titre (antiHB AB), characterized in that after M 20 measurement of the radioactivity on a first another carrier is contacted with unlabeled antigen-loaded carrier with another sample of the human body fluid, and the second carrier is incubated at a temperature between 18 and 45 ° C for 30 minutes to 15 hours, after which the second carrier is washed, introduced in a solution of radiolabeled antibody 25 and incubated for 30 minutes to 15 hours at a temperature between 18 and 45 ° C and washed the second time, after which the radioactivity of the second carrier is measured, whereby an increased number value compared with the first measurement indicates the presence of antigen, and a comparatively constant low number value compared to the first measurement indicates presence of antibody.